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Gentle Rhodamines for Live-Cell Fluorescence Microscopy.

Tianyan Liu1,2, Julian Kompa3, Jing Ling1,2

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New Gentle Rhodamines significantly reduce phototoxicity for long-term bioimaging. These cyclooctatetraene (COT) conjugated fluorophores enable advanced time-lapse applications with diminished cellular photodamage.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Microscopy

Background:

  • Rhodamine dyes are crucial for bioimaging but suffer from phototoxicity under prolonged illumination, limiting time-lapse studies.
  • Phototoxicity, particularly singlet-oxygen generation, is a major challenge for long-term live-cell imaging applications.
  • Existing rhodamine optimizations have largely overlooked the issue of phototoxicity.

Purpose of the Study:

  • To develop novel rhodamine fluorophores with reduced phototoxicity for enhanced time-lapse bioimaging.
  • To introduce a general strategy for creating "Gentle Rhodamines" that maintain excellent imaging properties while minimizing photodamage.
  • To create a versatile toolkit of Gentle Rhodamines for various cellular imaging targets and protein labeling.

Main Methods:

  • Conjugation of cyclooctatetraene (COT) to rhodamine scaffolds to create new fluorophores.
  • Evaluation of spectroscopic properties, cell permeability, and phototoxicity, including singlet-oxygen formation.
  • Application of COT-conjugated rhodamines in time-lapse imaging of mitochondria, DNA, actin, and HaloTag-labeled proteins.

Main Results:

  • Developed Gentle Rhodamines spanning the visible spectrum with significantly reduced phototoxicity and singlet-oxygen formation.
  • Demonstrated preserved spectroscopic properties and cell permeability compared to conventional rhodamines.
  • Showcased reduced cellular photodamage in time-lapse imaging of various cellular structures and protein labeling, enabling applications like voltage imaging.

Conclusions:

  • Gentle Rhodamines offer a viable solution to phototoxicity in long-term bioimaging.
  • The developed strategy provides a versatile toolkit for advanced time-lapse microscopy with reduced photodamage.
  • These new fluorophores expand the possibilities for live-cell imaging, including dynamic processes and voltage sensing.